Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein

• Published in: Molecular and biochemical parasitology 1999-06, Vol.101 (1), p.13-21
• Main Authors: Ruiz de Eguino, Arantxa D., Machı́n, Angeles, Casais, Rosa, Castro, Antonio M., Boga, José A., Martı́n-Alonso, José M., Parra, Francisco
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Animals; Antigens, Helminth - chemistry; binding sites; Blotting, Northern; calcium; Calcium - metabolism; Calcium-binding protein; calcium-binding proteins; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - immunology; Calcium-Binding Proteins - metabolism; Chromatography, Gel; cloning; Cloning, Molecular; complementary DNA; EF-hand; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; excretory-secretory products; Excretory–secretory antigen; Fasciola hepatica; Fasciola hepatica - chemistry; Fasciola hepatica - genetics; genbank/aj003822; gene expression; Genes, Helminth; Helix-loop-helix motif; Helminth Proteins; Humans; Immunoblotting; inorganic ions; Molecular Sequence Data; nucleotide sequences; Protein Conformation; Rabbits; recombinant DNA; recombinant proteins; Sequence Analysis, DNA; structural genes
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(99)00012-2

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory–secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory–secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca 2+-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.